There is significant interest in the use of analyte sensors based on quenched fluorescence. The list of analytes for which this type of sensor has been suggested includes oxygen, pH, CO2, and glucose. Such sensors have been used in connection with the various processes carried out in bio-reactors such as are utilized in the biotechnology, pharmaceutical and food and beverage industries (See e.g., Lakowicz, Principles of Fluorescence of Spectroscopy, 3rd edition, Springer 2006; R. Narayanaswamy, O. S. Wolfbeis (eds.): Optical Sensors: Industrial, Environmental and Diagnostic Applications, Analytical and Bioanalytical Chemistry, Springer (2004), 1618-2642; C. M. McDonagh et al., Characterization of porosity and response times of sol-gel-derived thin films for oxygen sensor applications, “J. Non Cryst. Solids, 306, (2002) 138-148.; P. A. Jorge et al, Application of quantum dots in optical fiber luminescent oxygen sensors, Applied Optics 45, 16, (2006), 3760-3764; and R. Jaaniso et al., Stability of luminescence decay parameters in oxygen sensitive polymer films doped with Pd-porphyrins, Proceedings of SPIE 5946, (2005), 59460L-1-59460L-10).
Initially, fluorescence based sensors monitored the quenching of fluorescent intensity in the time domain. However, many significant obstacles to reliable performance are encountered. A major issue which arises in using time domain measurement is that it requires sensing a change in the amplitude of the fluorescent signal. This change can be caused by a change in the concentration of the quenching agent (e.g., analyte), but also by a change in the incident power provided by the light source. Also, movement in the position of the fluorescent signal detector can cause an erroneous reading. Even more subtle issues like changes in the properties of the surface of the sensor material (e.g. roughness, index of refraction, and absorbance). Additionally, changes in the position or sensitivity of the photo-detector can lead to drift in the baseline reading.
The terms fluorophore, fluorescent dye, sensor dye and dye are used as equivalents in describing the present invention and are intended to connote an organic, organo-metallic or inorganic compound or mixture of compounds whose upper state fluorescent or phosphorescent lifetime is affected by a target analyte through collisional quenching or Forster type (e.g.: FRET) or dipole mediated energy exchange. In general, any light resulting from optical excitation is referred to as luminescent, whereas the term fluorescent light is frequently used to describe the light that results from singlet to singlet transitions, and the term phosphorescent is used to describe the light resulting from first order dis-allowed triplet to singlet transitions. As used herein the term fluorophore is intended to encompass both fluorescent and phosphorescent materials, and the term fluorescent signal is intended to also encompass a phosphorescent signal. Fluorophores which are capable of detecting a variety of analytes such as O2, pH, CO2 and glucose are known in the art. Recently, significant effort has focused on both actual sensor design and on optimization of fluorophore performance.
With advances in electronics and light sources, the use of information gathering in the frequency domain has become an attractive approach to the measurement of fluorescent signals. Sensors that utilize the phase delay of the fluorescence signal are based on fluorescence lifetime, which is a more intrinsic property of a fluorophore and therefore less likely to be affected by non-analyte induced changes in the environment adjacent to the fluorophore. Phase fluorometric systems work by detecting a change in the phase lag of the emitted fluorescent signal as a function of analyte concentration. In some cases it has been found to be a more efficacious basis for a sensor than monitoring the quenching of fluorescent intensity in the time domain. In general, a shorter (shorter than the emission) wavelength excitation source is modulated by a frequency, f, and a longer wavelength fluorescent signal is emitted at the same modulation frequency, but with a delay in phase. The phase delay is caused by the fact that the energy levels of the fluorescent material have finite time constants associated with them. In many ways, one can use a classical electrical analog to a low pass filter to understand the origin of the delay. The fluorescent states can be thought of as a capacitor which has a capacitance that is a function of the environment. At a given frequency, the phase of the signal passed by the low pass filter is mediated by the capacitor's value. In a similar way, the phase delay between the excitation signal and the emitted fluorescent signal is a function of the analyte concentration. An example of this delay is represented in FIG. 1. (See C. M. McDonagh et al., Phase fluorimetric dissolved oxygen sensor, Sensors and Actuators B 74, (2001) 124-130).
In FIG. 1 the excitation signal and the phase lagging fluorescent signal are shown. The relationship describing the phase delay, φ, and its relationship to the modulation frequency, f, and the fluorescent life time τ is also shown in FIG. 1. τ will change as the analyte concentration changes, which means that φ will likewise change as the analyte concentration changes. Methods and suitable data processing equipment which enable one to calculate the phase delay between the excitation signal and the fluorescence signal are known in the art and are described, for example, in Lakowicz, Principles of Fluorescence of Spectroscopy, 3rd edition, Springer 2006.
A significant problem that can occur with both intensity based and phase fluorimetric based analyte sensors, is photo-degradation of the fluorescent dye. Photo-degradation as used herein refers to the fact that the fluorescent lifetime of a fluorescent dye immobilized in a matrix can change due to extended exposure to light. The absorption of light by photo-sensitive fluorescent dyes over time can result in the formation of other photo-stable compounds. The electrons present in the photo-stable compounds are no longer able to contribute to the fluorescent nature of the dye, and thereby change the fundamental characteristics of the dye (See Sang-Kyung Lee and Ichiro Okura, Photostable Optical Oxygen Sensing Material: Platinum Tetrakis (pentafluorphenyl) porphryin Immobilized in Polystyrene, Analytical Communications, 34, (1997), 185-188; K. Oige et al, Effect of long-term aging oxygen sensitivity of luminescent Pd-tetrahenylporphrin/PMMA films, Sensors and Actuators B 106 (2005) 424-430.
Most currently used fluorophores absorb in the blue-green region of the visible spectrum. One significant consequence of this fact is that ambient light, especially sunlight, can change the material properties of the fluorophore and therefore significantly affect its behavior as a sensing element. If the rate of this change is rapid, the fluorophore's reliability as a sensing element will be adversely affected. Also, if the material properties of the fluorophore change during use, the baseline reading of the analyte concentration will likewise change erroneously during use. Such effects can lead to poor accuracy, low precision, and high drift. Especially for bioprocess applications, stringent performance requirements are placed on accuracy, precision and drift, as a result of which currently known fluorescent-based sensors do not fully meet many bioprocess requirements. Furthermore, such effects will make the storage requirements for fluorophores, especially those for use in disposable sensors, more onerous, as they will age during storage if exposed to ambient light.
In general, it is known that the rate at which changes to a fluorophore occur is directly proportional to the intensity of the excitation light and exposure to ambient light (as well as analyte concentration). One method of reducing deleterious effects is to reduce the exposure of the fluorescent dye to both stray light, and also to reduce the required amount of excitation light. The fluorescent dye can generally be relatively easily shielded from the stray light; however, reducing the required amount of excitation light is a much greater challenge. The present invention provides a major advance in terms of reducing the amount of required excitation light and avoiding degradation of the fluorophore while nevertheless ensuring that the amount of fluorescent signal which reaches the detector is sufficient
The construction of prior art fluorescence based sensors has generally favored using fiber-optic based illumination and collection geometries. Such a prior art design is shown in FIG. 2. In FIG. 2, 1 is the excitation LED, 2 is an optical filter which tailors the excitation spectrum such that it is matched to the absorption wavelength of the analyte sensitive dye 6. 3 is a fiber-optical coupler which allows the excitation light to travel into the common delivery/collection fiber 4, while allowing the fluorescent signal to simultaneously travel in the opposite direction. The fluorescent signal is then passed through optical filter 7 so that only the fluorescent signal reaches optical detector 8. 5 is a set of coupling optics (optional) to help increase collection and delivery of light from and to the dye, respectively. An obvious aspect of the system pictured in FIG. 2 is that the excitation light is remotely filtered and coupled into a single fiber, while the collected fluorescent signal is delivered to a photo-detector which is also located remotely from the dye. This allows a system where the optical sources and coupling all occur at the same location as the data processing electronics. While this simplifies some of the design and implementation issues and allows the use of fiber-optic for both delivery of the excitation light and collection of the fluorescence signal from a remote location, it is also limiting in several ways. First, the fiber's ability to withstand bending and other mechanical perturbation is limited. Leakage of both illumination light and signal light caused by bending the fiber or fiber bundle results in the excitation light actually impinging on the fluorophore being of lower than optimal power, and loss of the collected fluorescent signal can significantly reduce the signal to noise ratio. The use of multiple fibers or fiber bundles can help, but dramatically increases the cost and complexity of the system. The collection of the fluorescent signal is often the most vexing problem, and the ability of the fiber (or fiber bundle) to collect light is limited so that most systems of this type collect substantially less than 10% of the light emitted by the fluorophore. In fact, the maximum amount of the emitted fluorescence signal which any optic fiber based system can collect is substantially less than 50% unless the spot is deposited directly on the core of the fiber. The present invention overcomes the significant disadvantage of using optical fiber and enables the collection of greater than 50% and indeed can actually achieve almost 100% collection from an extended fluorescent source. Specifically by an “extended source”, we mean a source of greater than 1 mm in diameter that behaves as a Lambertian emitter. When the dye fluoresces it emits into all space, (i.e. over a hemisphere) so that approximately half of the light can be collected from the half plane. In general, a fluorescent dye layer that is thicker than a few tens of wavelengths will emit light as a Lambertian source into this half plane with an intensity that is proportional to Cos [θ] of the angle at which it is viewed, as is shown in FIG. 3. (See http://en.wikipedia.org/wiki/Image:LambertCosineLaw1.png).
In FIG. 3, I is the intensity, θ is the angle from the normal which the radiation is viewed, dA is a differential element of area, and dΩ is a differential element of solid angle. By looking at the emission pattern shown in FIG. 3, it is clear that a fiber's ability to collect light from such a source is limited. As the area of the source, A, increases, the fiber's collection efficiency is further reduced.
In general, fibers used in sensors have a circular collection area with a diameter of˜1 mm or even less. Additionally, the fibers can generally only collect light that hits the fiber with an angle˜≦±30 degrees. The definition of a Lambertian source, in contrast, stipulates that the emission angle is ±90 degrees, which is a mismatch to a typical fiber which can collect at most 30% of the emitted light, and frequently significantly less. It should also be noted that the fluorescent dyes are generally deposited in a 2-5 mm diameter circular spot which means that the 30% of light collected will be further reduced by another factor of 9 to 25, leading to a collection efficiency ranging from a little over 1% to at most 10%. Our invention, which utilizes free space transmission of the majority of at least one of the excitation light and the fluorescence signal (and preferably both) and in all cases avoids the use of fiber-optic, thereby achieves dramatically superior collection efficiency. As above-indicated, the present invention enables the collection of greater than 50% of the photons emitted by the fluorophore, and indeed can actually achieve almost 100% collection efficiency from extended fluorescent sources.
In order to quantify the mismatch between the Lambertian emission of a fluorescent dye and the collection efficiency of a typical optical fiber as compared to the collection efficiency achievable by the present invention, it is necessary to consider a concept referred to as brightness. The quantity shown in Equation 1, which is called brightness, can be shown from fundamental thermodynamics to be a conserved quantity. (See Ross, McCluney, Introduction to Radiometry and Photometry).
                    Power                  Solid          ⁢                                          ⁢          Angle          ×          Area                                    Equation        ⁢                                  ⁢        1            
The denominator, which is the product of the solid angle radiated to/from and the area radiated to/from, is called étendue. This concept means that it is never possible to take a source having a given power and étendue and make it brighter. Likewise, it is not possible to couple into a smaller area or smaller angular acceptance without losing power. With the concept of conservation of brightness in mind, consider a fluorescent source having a 5 mm diameter (area=π(2.5)2 mm2) radiating into a half plane. Equation 1 tells us that the majority of the radiated light cannot be collected by a fiber with a 1 mm diameter (area=π(0.5)2 mm2) and one third of the collection solid angle. Taking the ratio of the area of the 5 mm diameter fluorescent spot to the area of the 1 mm diameter fiber we have:
                                          emission            ⁢                                                  ⁢            area                                collection            ⁢                                                  ⁢            area                          =                                            π              ⁢                                                          ⁢                              2.5                2                                                    π              ⁢                                                          ⁢                              0.5                2                                              =          25                                    Equation        ⁢                                  ⁢        2            
Equation 2 shows that the area factor alone in Equation 1 causes a decrease in collection efficiency by a factor of 25 for a typical fiber. If one also accounts for the angular distribution of the Lambertian source emitting into a half sphere compared to the fiber's angular limited collection ability, we have a source which has an étendue that is approximately 75 times that of the fiber. It would be exactly a factor of 75 only if the extended source were uniform and not Lambertian with the same solid angle limits. In order for brightness to be conserved, and assuming a uniform illumination source, the difference will be made up by a factor of 75 loss in power coupled into the optical fiber. If the resulting signal after the photo-detector does not result in a sufficient signal to noise ratio in the detection electronics, the excitation source power will need to be increased accordingly, or the sensor will not accurately measure the phase angle. This increased excitation power, in turn, results in an increased rate of photo-degradation of the dye. Additionally, if the collection fiber is bent beyond its ability to guide light so that there is leakage of the signal, the excitation light will also need to be increased to make up for this loss. If the loss in fluorescent signal power causes a significant decrease in the signal to noise ratio, the increased illumination power results in a significantly increased rate of photo-degradation of the fluorophore. Our design does not use fibers, so it is not susceptible to this issue. Additionally, because of the enhanced collection efficiency of the apparatus of the present invention, in many instances we are able to effectively utilize excitation light sources which deliver intensities<1 μW/mm2 and not realize the same photo-degradation rate provided by a fiber based source delivering˜10 μW/mm2.